PROJECT SUMMARY Devastating influenza A virus (IAV) pandemics arise when non-human adapted strains overcome human species barriers to successfully infect and sustain human-to-human transmission. The novelty of a pandemic strain poses a substantial burden to human health leading to increased incidences of severe respiratory complications, hospitalizations, and death when compared to seasonal influenza. The most recent pandemic in 2009 (pH1N1) arose from a reassortant swine IAV that contained human, swine, and avian adapted gene segments. To understand how these pandemic IAVs overcome species barriers, we must understand the mechanisms that lead to viral adaptation to human hosts. Recent work in our lab has demonstrated that regulation of gene expression from the IAV M segment plays a role in host adaptation. The IAV M segment encodes two major proteins by alternative splicing. Matrix 1 (M1) gives the virion its structure and is encoded by the colinear mRNA, and M2, which is needed for viral replication, is encoded by a splice product of the M1 mRNA. We have used reverse genetics to engineer recombinant viruses that share seven of the eight IAV gene segments but differ in the origin of the M segment, allowing us to evaluate properties of this segment. Recombinant viruses carrying avian or human adapted M segments replicate to similar titers and express similar levels of M1 and M2 protein when infecting avian cells. However, in human cells, the avian M segment virus replicates less efficiently and expresses significantly higher levels of M2 mRNA and protein than the human M segment virus. Mammalian adapted M segments maintain low levels of M2 in human cells. These data lead to my hypothesis that, following introduction into mammalian hosts, mutations to the avian IAV M segment are needed to re- establish optimal virus-host interactions important for regulation of M2 expression. In Aim 1, I will map the M sequence determinants that underly differential M gene expression between avian and mammalian adapted M segments. I have used reverse genetics to generate avian M segment viruses that carry nucleotide mutations identified in the pH1N1 M segment. I will infect human cells with these viruses. Then I will evaluate the impact of these mutations on avian and human adapted M1 and M2 mRNA and protein steady state levels and stability. In Aim 2, I will investigate the role of host factors in differential M2 expression between avian and human adapted M segments. I will determine the binding affinities of avian and human M1 mRNAs with host splicing factors using biochemical methods and evaluate subcellular localization of M1 and M2 mRNAs during infection by microscopy. Through these experiments, I will elucidate the molecular mechanisms that underly the species specificity IAV M segment gene expression. These studies will reveal additional mechanisms of IAV adaptation to human hosts, which could be used to better assess the pandemic risks of future IAV zoonoses.